Diverse Pulsed-field Gel Electrophoresis Research Articles

Prevalence and Characterization of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolated from Dogs and Cats in South Korea.

Overall, 836 Escherichia coli isolates (695 isolates from dogs and 141 from cats) were recovered from the diarrhea, skin/ear, urine, and genitals of dogs and cats between 2018 and 2019. Cefovecin and enrofloxacin resistance were noted in 17.1% and 21.2% of E. coli isolates, respectively. The cefovecin and enrofloxacin resistance rates were higher in dog isolates (18.1% and 22.9%) compared with the rates in cat isolates (12.1%, 12.8%). Interestingly, resistance to both antimicrobials was noted in 10.8% (90/836) of the isolates, predominantly in isolates from dogs. blaCTX-M-14, blaCTX-M-15, and blaCMY-2 were the most frequent extended-spectrum β-lactamase/plasmid-mediated AmpC β-lactamase (ESBL/AmpC)- gene types. The co-existence of blaCTX-M andblaCMY-2 was noted in six E. coli isolates from dogs. Sequencing analysis demonstrated that S83L and D87N in gyrA and S80I in parC were the most frequent point mutations in the quinolone resistance-determining regions of the cefovecin and enrofloxacin-resistant isolates. A total of 11 isolates from dogs carried the plasmid-mediated quinolone resistance genes (six aac(6')-Ib-cr, four qnrS, and one qnrB), while only two cat isolates carried the qnrS gene. Multilocus sequence typing of the cefovecin and enrofloxacin-resistant isolates revealed that sequence type (ST)131 E. coli carrying blaCTX-M-14 and blaCTX-M-15 genes and ST405 E. coli carrying blaCMY-2 gene were predominant among the isolated E. coli strains. The majority of the ESBL/AmpC-producing isolates displayed diverse pulsed-field gel electrophoresis profiles. This study demonstrated that third-generation cephalosporin- and fluoroquinolone-resistant E. coli were widely distributed in companion animals. The detection of the pandemic ST131 clone carrying blaCTX-M-14/15 in companion animals presented a public health threat.

Prevalence and Genomic Characteristics of mcr-Positive Escherichia coli Strains Isolated from Humans, Pigs, and Foods in China.

Colistin is one of the last-resort antibiotics for treating infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, mcr genes conferring resistance to colistin have been widely identified, which is considered a global threat to public health. Here, we investigated the prevalence and characteristics of mcr-harboring Escherichia coli strains isolated from humans, animals, and foods in China by PCR, antimicrobial susceptibility testing, conjugation experiments, molecular typing, genome sequencing, and bioinformatics analysis. In total, 135 mcr-1-harboring E. coli isolates were acquired from 847 samples, and 6 isolates carried mcr-3. Among them, 131 isolates were MDR bacteria. Sixty-five resistance genes conferring resistance to multiple antimicrobials were identified in 135 isolates. The diverse pulsed-field gel electrophoresis (PFGE) patterns and sequence types (STs) of mcr-1-carrying isolates demonstrated that clonal dissemination was not the dominant mode of mcr-1 transmission. Seven types of plasmids were able to carry mcr-1 in this study, including IncI2, IncX4, IncHI2, p0111, IncY, and two hybrid plasmids. The genetic structures carrying mcr-1 of 60 isolates were successfully transferred into the recipient, including 25 IncI2 plasmids, 23 IncX4 plasmids, and an IncHI2 plasmid. mcr-1-pap2 was the dominant mcr-1-bearing structure, followed by ISApl1-mcr-1-pap2-ISApl1 (Tn6330) and ISApl1-mcr-1-pap2, among 7 mcr-1-bearing structures of 135 isolates. In conclusion, IncI2, IncX4, and IncHI2 plasmids were the major vectors spreading mcr-1 from different geographical locations and sources. The prevalence of Tn6330 may accelerate the transmission of mcr-1. Continuous surveillance of mcr-1 and variants in bacteria is vital for evaluating the public health risk posed by mcr genes. IMPORTANCE The spread of polymyxin-resistant Enterobacteriaceae poses a significant threat to public health and challenges the therapeutic options for treating infections on a global level. In this study, mcr-1-bearing ST10 E. coli was isolated from pigs, pork, and humans simultaneously, which demonstrated that ST10 E. coli was an important vehicle for the spread of mcr-1 among animals, foods, and humans. The high prevalence of mcr-1-positive E. coli strains in pigs and pork and the horizontal transmission of mcr-1-bearing plasmids in diverse E. coli strains suggest that pigs and pork are important sources of mcr-1-positive strains in humans and pose a potential threat to public health. Additional research on the prevalence and characteristics of mcr-1-positive E. coli is still required to facilitate early warning to improve polymyxin management in hospitals.

Phenotypic and genotypic characterization of Listeria monocytogenes in clinical ruminant cases in Korea

Listeria monocytogenes, a foodborne human and veterinary pathogen, is associated with high mortality rates in ruminants. However, no studies have investigated the antimicrobial resistance of L. monocytogenes isolates from clinical ruminant cases. This study aimed to determine the phenotypic and genotypic characteristics of L. monocytogenes isolates from clinical cases of Korean ruminants. We collected 24 L. monocytogenes isolates from aborted bovine fetuses and goats presenting with listeriosis-related symptoms. The isolates were subjected to PCR serogrouping, conventional serotyping, virulence gene detection, and antimicrobial susceptibility testing. Furthermore, pulsed-field gel electrophoresis and multilocus sequence typing were used to classify and compare genetic diversity among the isolates, including human L. monocytogenes isolates. The most prevalent L. monocytogenes serotypes were 4b (Ⅳb), 1/2a (Ⅱa; Ⅱc), and 1/2b (Ⅱb). All isolates harbored the virulence genes; however, llsX-encoding listeriolysin were identified only in serotypes 4b and 1/2b. All isolates, including two found in humans, formed three genetically diverse pulsed-field gel electrophoresis clusters according to serotype, lineage, and sequence type. The most prevalent sequence type was ST1, followed by ST365 and ST91. The isolates from ruminants with listeriosis were resistant to oxacillin and ceftriaxone and showed diverse lineage, serotype (serogroup), and sequence type characteristics. Considering that the atypical sequence types exhibited clinical manifestations and histopathological lesions, further study is needed to elucidate the pathogenicity of genetically diverse ruminant L. monocytogenes isolates. Furthermore, continuous monitoring of antimicrobial resistance is required to prevent the emergence of L. monocytogenes strains resistant to common antimicrobials.

Prevalence of qnrS-positive Escherichia coli from chicken in Thailand and possible co-selection of isolates with plasmids carrying qnrS and trimethoprim-resistance genes under farm use of trimethoprim

One hundred and twenty chicken samples from feces (n = 80), the carcass surface at slaughter at 2 meat chicken farms (n = 20), and retail chicken meat from 5 markets (n = 20) collected during 2018 and 2019 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) in Escherichia coli. We detected qnrS-positive E. coli in a total of 74 samples from feces (n = 59), the carcass surface (n = 7), and retail meat (n = 8). These 74 qnrS-positive isolates were tested for antimicrobial susceptibility to determine the minimum inhibitory concentrations (MICs) of certain antimicrobials and genetically characterized. Ampicillin-resistance accounted for 71 of the 74 isolates (96%), followed by resistance to oxytetracycline (57/74; 77%), enrofloxacin (ERFX) (56/74; 76%), sulfisoxazole (SUL) (56/74; 76%), trimethoprim (TMP) (49/74; 66%), and dihydrostreptomycin (48/74; 65%). All farm-borne SUL- and TMP-resistant isolates except one were obtained from samples from farm A where a combination of sulfadiazine and TMP was administered to the chickens. Concentrations of ERFX at which 50 and 90% of isolates were inhibited were 2 μg/mL and 32 μg/mL, respectively. Diverse pulsed-field gel electrophoresis (PFGE) patterns of XbaI-digested genomic DNA were observed in the qnrS-positive isolates from fecal samples. Several isolates from feces and the carcass surface had identical XbaI-digested PFGE patterns. S1-nuclease PFGE and Southern blot analysis demonstrated that 7 of 11 dfrA13-positive fecal isolates carried both the qnrS and dfrA13 genes on the same plasmid, and 2 of 3 dfrA1-positive isolates similarly carried both qnrS and dfrA1 on the same plasmid, although the PFGE patterns of XbaI-digested genomic DNA of the isolates were different. These results suggest that the qnrS gene is prevalent in chicken farms via horizontal transfer of plasmids and may partly be co-selected under the use of TMP.

A prolonged multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales due to horizontal transmission of the IncN plasmid

A multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales (IMP-6-CPE) occurred at an acute care hospital in Japan. This study was conducted to understand the mechanisms of IMP-6-CPE transmission by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing and whole-genome sequencing (WGS), and identify risk factors for IMP-6-CPE acquisition in patients who underwent abdominal surgery. Between July 2013 and March 2014, 22 hospitalized patients infected or colonized with IMP-6-CPE (Escherichia coli [n = 8], Klebsiella oxytoca [n = 5], Enterobacter cloacae [n = 5], Klebsiella pneumoniae [n = 3] and Klebsiella aerogenes [n = 1]) were identified. There were diverse PFGE profiles and sequence types (STs) in most of the species except for K. oxytoca. All isolates of K. oxytoca belonged to ST29 with similar PFGE profiles, suggesting their clonal transmission. Plasmid analysis by WGS revealed that all 22 isolates but one shared a ca. 50-kb IncN plasmid backbone with blaIMP-6 suggesting interspecies gene transmission, and typing of plasmids explained epidemiological links among cases. A case-control study showed pancreatoduodenectomy, changing drains in fluoroscopy room, continuous peritoneal lavage and enteric fistula were associated with IMP-6-CPE acquisition among the patients. Plasmid analysis of isolates in an outbreak of IMP-6-CPE suggested interspecies gene transmission and helped to clarify hidden epidemiological links between cases.

Molecular typing, virulence genes and drug resistance of Salmonella typhimurium in Longyan city

Objective To analyze the molecular typing characteristics by pulsed field gel electrophoresis (PFGE), drug resistance and virulence genes of Salmonella typhimurium in Longyan city in order to provide reference for the prevention and control. Methods A total of 79 Salmonella typhimurium strains were isolated from sporadic cases of diarrhea and food poisoning and raw poultry meat samples during 2010 to 2017. PFGE was performed to measure the minimum inhibitory concentrations (MIC) of 15 commonly used drugs against them. PCR was used to detect nine virulence genes (sopB, invA, sifA, sscA, sseE, spvB, pefA, spvR, spvC) in 55 strains. Results The 79 Salmonella typhimurium strains belonged to 61 PFGE types. There were 10, three and four strains of P1, P3 and P21 types, respectively. Seven P1 type strains were isolate from one food poisoning event. According to the 85% classification standard, 79 Salmonella typhimurium strains could be divided into five predominant gene clusters (G1-G5). Drug susceptibility test showed that the 79 strains had the highest resistance rate to ampicillin (88.61%), followed by that to tetracycline (87.34%) and streptomycin (73.41%). Multidrug resistant bacteria resistant to three or more antibacterial drugs accounted for 84.81% (67/79). All of the 55 strains carried invA, sopB, sseE and sscA genes. The other five genes, sifA, spvC, spvB, spvR and pefA, were detected in 54, 31. 10, 11 and 12 strains, respectively. There were 76.4% (42/55) of the strains carrying five or six virulence genes and all were positive for invA, sopB, sseE, sscA and sifA, and negative for spvB, spvR and pefA. The strains carrying all of the nine virulence genes accounted for 18.2% (10/55). Conclusions Salmonella typhimurium strains isolated in Longyan city had a diverse PFGE type. P1, P3 and P21 types were the three predominant PFGE types. In the food poisoning event, PFGE molecular typing could quickly alert the outbreak and traceability of Salmonella typhimurium. Attention should be paid to the multidrug resistance in Salmonella typhimurium. Monitoring of multidrug-resistant strains and supervision on antibacterial drug usage should be strengthened. Salmonella typhimurium had high virulence as it carried many virulence genes. Key words: Salmonella typhimurium; PFGE molecular typing; Cluster analysis; Minimum inhibitory concentration; Multidrug resistance; Virulence gene; Salmonella virulence island; Salmonella plasmid virulence gene

Prevalence of meticillin-resistant and -susceptible coagulase-negative staphylococci with the first detection of the mecC gene among cows, humans and manure in Tunisia

In Europe, a novel mecA homologue - mecC (formerly mecALGA251)- has emerged recently in staphylococci from animals, humans and the environment. This paper reports the first occurrence of the mecC gene in Staphylococcus sciuri from cows and manure in Tunisia and Africa. Forty-nine coagulase-negative staphylococci (CNS) were isolated from the milk of cows with mastitis (n=20), manure (n=20) and human nares (n=9), including 16 Staphylococcus equoruim (32.6%), 12 S. xylosus (24.5%), 12 S. sciuri (24.5%), two S. saprophyticus (4.1%), two S. haemolyticus (4.1%), two S. lentus (4.1%), two S. vitulinus (4.1%) and one S. cohnii (2%). CNS from the three origins carried various resistance genes [mecA, blaZ, tet(K), erm(A), erm(B), msr(A)], suggesting an ongoing genetic exchange among CNS from the three niches. The mecA gene was detected in CNS (n=11) recovered from cows, manure and humans, whereas the mecC gene (n=3) was only detected in CNS from cows and manure. Various staphylococcal cassette chromosome mec (SCCmec) - SCCmec type I (n=1), II (n=3), IV (n=2), V/VII (n=2) and untypeable (n=3) - and diverse pulsed-field gel electrophoresis (PFGE) patterns were observed in mecA-positive CNS. Otherwise, similar SCCmec types and PFGE patterns were found in meticillin-resistant CNS within different farms and origins, showing the potential of SCCmec interspecies exchange and circulation of the same clones of meticillin-resistant CNS in the human-animal-environment interface. This study provides baseline data to support clonal dissemination of CNS between cows, humans and manure, and indicates the possible transmission of the mecC gene to humans in contact with cows and manure.

Resistance mechanisms and molecular epidemiology of chlorhexidine acetate-resistant Klebsiella pneumoniae clinical isolates

Objective To investigate the chlorhexidine acetate-resistance in Klebsiella pneumoniae (K.pneumoniae) clinical isolates and to analyze the possible mechanisms and molecular epidemiology of resistant isolates. Methods A total of 332 K. pneumoniae clinical isolates were collected in the First Affiliated Hospital of Wenzhou Medical University in 2015. Standard agar dilution was used to screen chlorhexidine acetate-resistant isolates. The minimum inhibition concentrations (MIC) of chlorhexidine acetate to resistant isolates with and without the presence of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which was an efflux pump inhibitor, were analyzed. Efflux pump genes of cepA, qacE and qacΔE1 that carried by and expressed in those isolates were detected by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-qPCR), respectively. The biofilm formation ability was measured by crystal violet staining. The homology among the chlorhexidine acetate-resistant isolates was investigated with multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Results Twenty-five K. pneumoniae strains were resistant to chlorhexidine acetate. The MIC values of chlorhexidine acetate for them were reduced by at least four-fold in the presence of CCCP. Strains carrying the genes of cepA, qacE and qacΔE1 accounted for 100%, 40% and 40%, respectively. The expression of the efflux pump genes in the chlorhexidine acetate-resistant isolates was higher than that in the susceptible isolates. The biofilm formation ability of the chlorhexidine acetate-resistant isolates was better than that of the susceptible isolates. Furthermore, negative, weak-positive and positive biofilm formation ability was observed in four (16%), 20 (80%) and one (4%) strains, respectively. The results of MLST and PFGE showed that the 25 chlorhexidine acetate-resistant isolates belonged to 19 different sequence types (ST) with diverse PFGE patterns. Conclusions This study suggested that active efflux was the main mechanism of chlorhexidine acetate resistance in K. pneumoniae. The 25 chlorhexidine acetate-resistant K. pneumoniae strains possessed different biofilm formation ability and shared low homology. Key words: Chlorhexidine acetate; Klebsiella pneumoniae; Resistance mechanism; Molecular epidemiology

Diversity of metallo-β-lactamase-encoding genes found in distinct species of Acinetobacter isolated from the Brazilian Amazon Region.

BACKGROUND The multidrug resistance (MDR) phenotype is frequently observed in Acinetobacter baumannii, the most clinically relevant pathogenic species of its genus; recently, other species belonging to the A. calcoaceticus-A. baumannii complex have emerged as important MDR nosocomial pathogens.OBJECTIVES The present study aimed to verify the occurrence of metallo-β-lactamase genes among distinct Acinetobacter species in a hospital located in the Brazilian Amazon Region.METHODS Antimicrobial susceptibility profiles were determined by broth microdilution. The genetic relationships among these isolates were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Pyrosequencing reads of plasmids carrying the bla NDM-1 gene were generated using the Ion Torrent™ platform sequencing.FINDINGS A total of six isolates carried bla NDM-1: A. baumannii (n = 2), A. nosocomialis (n = 3), and A. pittii (n = 1); three carried bla IMP-1: A. baumannii, A. nosocomialis, and A. bereziniae. Resistance to colistin was observed for an NDM-1-producing A. nosocomialis isolate. Diverse PFGE patterns and sequence types were found among A. nosocomialis and A. baumannii isolates. The bla NDM-1 sequence was inserted in a Tn125 transposon, while the bla IMP-1 was found as a gene cassette of the class 1 integron In86.MAIN CONCLUSIONS To the best of our knowledge, this is the first report describing the dissemination of bla NDM-1 among distinct Acinetobacter species recovered from the same hospital in South America.

High carriage rate of mcr-1 and antimicrobial resistance profiles of mcr-1-positive Escherichia coli isolates in swine faecal samples collected from eighteen provinces in China

The mcr-1 gene is a plasmid-borne and globally distributed colistin-resistance gene, which threatens the last-line role of colistin in treatment of infections caused by multi-drug resistant(MDR) Gram-negative bacteria in humans. The aim of this study was to obtain a comprehensive understanding of mcr-1 prevalence in the pig industry in China. A total of 600 faecal samples were collected from 60 swine farms in 18 provinces of China. Faecal DNA was extracted and subjected to PCR screening to detect the presence of mcr-1. Positive samples were randomly selected for isolation of colistin-resistant bacteria. A total of 152 mcr-1-positive isolates collected from different provinces were characterized by antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) typing. Extremely high rate of faecal carriage of mcr-1(457/600, 76.2%) was observed, with positive rates ranging from 45.0%–100% among the provinces. All colistin-resistant colonies were identified as Escherichia coli and mcr-1-positive. The 152 representative mcr-1-positive E. coli isolates showed high level resistance to ampicillin (79.60%), tetracycline(94.74%), doxycycline(93.42%), florfenicol(78.29%), enrofloxacin(44.08%), and ciprofloxacin(38.82%) and low level resistance to ceftiofur(26.32%), cefoxitin(0.66%), and amikacin(0.66%). No resistant isolates were detected for gentamicin, meropenem, and tigecycline. The mcr-1-positive E. coli strains exhibited highly diverse PFGE patterns, which suggested that horizontal transfer through plasmids or other mobile elements was the main mechanism for the wide dissemination of mcr-1 in pig farms. Similar studies are warranted to continuously monitor colistin resistance and mcr-1 prevalence in food-animal production.

Prevalence and Molecular Characterization of mcr-1-Positive Salmonella Strains Recovered from Clinical Specimens in China.

The recently discovered colistin resistance element, mcr-1, adds to the list of antimicrobial resistance genes that rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics but also the last-line agents of carbapenems and colistin. This study investigated the prevalence of the mobile colistin resistance determinant mcr-1 in Salmonella strains recovered from clinical settings in China and the transmission potential of mcr-1-bearing mobile elements harbored by such isolates. The mcr-1 gene was recoverable in 1.4% of clinical isolates tested, with the majority of them belonging to Salmonella enterica serotype Typhimurium. These isolates exhibited diverse pulsed-field gel electrophoresis (PFGE) profiles and high resistance to antibiotics other than colistin and particularly to cephalosporins. Plasmid analysis showed that mcr-1 was carried on a variety of plasmids with sizes ranging from ∼30 to ∼250 kb, among which there were conjugative plasmids of ∼30 kb, ∼60 kb, and ∼250 kb and nonconjugative plasmids of ∼140 kb, ∼180 kb, and ∼240 kb. Sequencing of representative mcr-1-carrying plasmids revealed that all conjugative plasmids belonged to the IncX4, IncI2, and IncHI2 types and were highly similar to the corresponding types of plasmids reported previously. Nonconjugative plasmids all belonged to the IncHI2 type, and the nontransferability of these plasmids was attributed to the loss of a region carrying partial or complete tra genes. Our data revealed that, similar to the situation in Escherichia coli, mcr-1 transmission in Salmonella was accelerated by various plasmids, suggesting that transmission of mcr-1-carrying plasmids between different species of Enterobacteriaceae may be a common event.

Molecular Typing of Fluoroquinolone-Resistant Campylobacter jejuni Isolated from Broilers in Japan Using Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis.

Fluoroquinolone-resistant Campylobacter jejuni isolates from broilers in Japan were characterized using multilocus sequence typing and pulsed-field gel electrophoresis (PFGE) in order to elucidate the genetic relationship between these strains. Forty-three of the isolates were classified into 20 sequence types and were clustered into 21 PFGE types with 70% similarity. The most dominant clonal complex (CC) was CC-21 (41.9%). Diverse PFGE patterns were observed within the same CC, but the combined analysis of PFGE type and CC revealed that the strains with the same combination were isolated from the same district or neighboring districts. On the other hand, strains with the same combination pattern were also isolated from geographically distant districts. Our results elucidate two possible reasons for the prevalence of fluoroquinolone-resistant C. jejuni among broiler farms: (1) the resistant C. jejuni is clonally disseminated within the limited area, and (2) susceptible C. jejuni acquired fluoroquinolone resistance during the use of fluoroquinolone on the farms.

Dissemination and Characterization of Plasmids Carrying oqxAB-blaCTX-M Genes in Escherichia coli Isolates from Food-Producing Animals

BackgroundThe association of PMQR and ESBLs in negative-bacteria isolates has been of great concern. The present study was performed to investigate the prevalence of co-transferability of oqxAB and bla CTX-M genes among the 696 Escherichia coli (E. coli) isolates from food-producing animals in South China, and to characterize these plasmids.MethodsThe ESBL-encoding genes (bla CTX-M, bla TEM and bla SHV), and PMQR (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6’)-Ib-cr, qepA, and oqxAB) of these 696 isolates were determined by PCR and sequenced directionally. Conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blotting experiments were performed to investigate the co-transferability and location of oqxAB and bla CTX-M. The EcoRI digestion profiles of the plasmids with oqxAB-bla CTX-M were also analyzed. The clonal relatedness was investigated by PFGE.ResultsOf the 696 isolates, 429 harbored at least one PMQR gene, with oqxAB (328) being the most common type; 191 carried bla CTX-M, with bla CTX-M-14 the most common. We observed a significant higher prevalence of bla CTX-M among the oqxAB-positive isolates (38.7%) than that (17.4%) in the oqxAB-negative isolates. Co-transferability of oqxAB and bla CTX-M was found in 18 of the 127 isolates carrying oqxAB-bla CTX-M. These two genes were located on the same plasmid in all the 18 isolates, with floR being on these plasmids in 13 isolates. The co-dissemination of these genes was mainly mediated by F33:A-: B- and HI2 plasmids with highly similar EcoRI digestion profiles. Diverse PFGE patterns indicated the high prevalence of oqxAB was not caused by clonal dissemination.Conclusion bla CTX-M was highly prevalent among the oqxAB-positive isolates. The co-dissemination of oqxAB-bla CTX-M genes in E. coli isolates from food-producing animals is mediated mainly by similar F33:A-: B- and HI2 plasmids. This is the first report of the co-existence of oqxAB, bla CTX-M, and floR on the same plasmids in E. coli.

Multiple Antibiotic Resistance of Vibrio cholerae Serogroup O139 in China from 1993 to 2009

Regarded as an emerging diarrheal micropathogen, Vibrio cholerae serogroup O139 was first identified in 1992 and has become an important cause of cholera epidemics over the last two decades. O139 strains have been continually isolated since O139 cholera appeared in China in 1993, from sporadic cases and dispersed foodborne outbreaks, which are the common epidemic types of O139 cholera in China. Antibiotic resistance profiles of these epidemic strains are required for development of clinical treatments, epidemiological studies and disease control. In this study, a comprehensive investigation of the antibiotic resistance of V. cholerae O139 strains isolated in China from 1993 to 2009 was conducted. The initial O139 isolates were resistant to streptomycin, trimethoprim-sulfamethoxazole and polymyxin B only, while multidrug resistance increased suddenly and became common in strains isolated after 1998. Different resistance profiles were observed in the isolates from different years. In contrast, most V. cholerae O1 strains isolated in the same period were much less resistant to these antibiotics and no obvious multidrug resistance patterns were detected. Most of the non-toxigenic strains isolated from the environment and seafood were resistant to four antibiotics or fewer, although a few multidrug resistant strains were also identified. These toxigenic O139 strains exhibited a high prevalence of the class I integron and the SXT element, which were rare in the non-toxigenic strains. Molecular subtyping of O139 strains showed highly diverse pulsed-field gel electrophoresis patterns, which may correspond to the epidemic state of sporadic cases and small-scale outbreaks and complex resistance patterns. Severe multidrug resistance, even resistance transfers based on mobile antibiotic resistance elements, increases the probability of O139 cholera as a threat to public health. Therefore, continual epidemiological and antibiotic sensitivity surveillance should focus on the occurrence of multidrug resistance and frequent microbial population shifts in O139 strains.

Comparison of genetic epidemiology of vancomycin-resistant Enterococcus faecium isolates from humans and poultry

This study was conducted to investigate the molecular characteristics and genetic relatedness of vancomycin-resistant Enterococcus faecium (VREF) isolates obtained from humans and poultry in Korea. A total of 147 VREF isolates from humans (71 clinical isolates) and poultry (76 isolates) in Korea were compared with respect to their antibiotic susceptibilities, organization of the Tn1546 transposon element, detection of virulence genes (esp and hyl), pulsed-field gel electrophoresis (PFGE) typing and multilocus sequence typing (MLST). All of the human and poultry isolates had the vanA gene and 11.3% (8/71) of the clinical isolates showed the VanB phenotype/vanA genotype. PCR mapping of the Tn1546 elements was different for isolates of the two groups: human isolates were classified into five transposon types, whereas all poultry isolates were identical to Tn1546 of E. faecium strain BM4147. The esp gene was detected in both human (93.0%, 66/71) and poultry (26.3%, 20/76) isolates, as was the hyl gene (human isolates: 80.3%, 57/71; poultry isolates: 26.3%, 20/76). Using MLST, the 71 human isolates could be divided into ten sequence types (STs) belonging to clonal complex (CC) 17 (except for one singleton). The 76 poultry isolates were categorized into 14 STs and 88.2% (67/76) of the poultry isolates belonged to CC26. PFGE typing of the human isolates demonstrated diverse PFGE profiles among the strains. However, the PFGE patterns of the poultry isolates were possibly related to the strains collected from individual farms. These data suggest that epidemic clonal groups of human and poultry VREFs in Korea have evolved through different evolutionary processes.

Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis

The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes.

Polyclonal Diffusion of Beta-Lactamase-Producing Enterococcus faecium

We describe here the isolation of 8 beta-lactamase-producing multidrug-resistant Enterococcus faecium isolates in 2010. All strains showed diverse pulsed-field gel electrophoresis (PFGE) profiles, all belonging to the same clonal complex, CC17. By PCR and hybridization experiments, the entire blaZ-blaI-blaR1 operon was found. The beta-lactamase activity was demonstrated at a high inoculum and in the presence of methicillin after overnight incubation.

Comparison of Two Multilocus Variable-Number Tandem-Repeat Methods and Pulsed-Field Gel Electrophoresis for Differentiating Highly Clonal Methicillin-Resistant Staphylococcus aureus Isolates

In the United Kingdom, EMRSA-15 and EMRSA-16 account for the majority (∼90%) of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections. Currently, the standard typing technique, pulsed-field gel electrophoresis (PFGE), is laborious and insufficient for discriminating between closely related subtypes of EMRSA-15 and -16. The objective of the present study was to compare the usefulness of multilocus variable-number tandem-repeat fingerprinting (MLVF) and multilocus variable-number tandem-repeat analysis (MLVA) with PFGE for subtyping these highly clonal MRSA lineages. A panel of 85 MRSA isolates (41 EMRSA-15, 20 EMRSA-16, and 24 MRSA isolates with diverse PFGE patterns) was investigated. In addition, a further 29 EMRSA-15s with identical PFGE patterns from two geographically linked but epidemiologically distinct outbreaks and several sporadic cases were analyzed. PFGE, MLVF, and MLVA resolved 66 (Simpson's index of diversity [SID] = 0.984), 51 (SID = 0.95), and 42 (SID = 0.881) types, respectively, among the 85 MRSA isolates. MLVF was more discriminatory than MLVA for EMRSA-15 and -16 strains, but both methods had comparable discriminatory powers for distinguishing isolates in the group containing diverse PFGE types. MLVF was comparable to PFGE for resolving the EMRSA-15s but had a lower discriminatory power for the EMRSA-16s. MLVF and MLVA resolved the 29 isolates with identical PFGE patterns into seven and six subtypes, respectively. Importantly, both assays indicated that the two geographically related outbreaks were caused by distinct subtypes of EMRSA-15. Taken together, the data suggest that both methods are suitable for identifying and tracking specific subtypes of otherwise-indistinguishable MRSA. However, due to its greater discriminatory power, MLVF would be the most suitable alternative to PFGE for hospital outbreak investigations.

Intermediately virulent Rhodococcus equi isolates from pigs in Slovenia: discovery of new plasmid types and assessment of genetic diversity by pulsed-field gel electrophoresis

The presence of large plasmids in 30 <I>Rhodococcus equi</I> strains isolated from pig lymph nodes with granulomatous changes was investigated. Plasmid DNAs were isolated and digested with the restriction endonucleases <I>Bam</I>HI, <I>Eco</I>RI, <I>Eco</I>T22I and <I>Hind</I>III for detailed comparison and estimation of plasmid sizes. A total of nine isolates were identified as intermediately virulent (VapB-positive), harbouring large plasmids of type 5 (<I>n</I> = 5) and four new variants that we tentatively designated as type 19 (<I>n</I> = 1), 20 (<I>n</I> = 1), 21 (<I>n</I> = 1) and 24 (<I>n</I> = 1). All isolates were subjected to genotyping with pulsed-field gel electrophoresis (PFGE). High genetic diversity was observed: 21 distinct genotypes were detected; five were found in multiple isolates and the others were unique. Isolates of the same plasmid type exhibited different PFGE profiles and vice versa. In a few cases, multiple strains from certain farms were analysed, the majority of which exhibited diverse PFGE profiles. Our findings demonstrate the presence of a wide variety of <I>R. equi</I> strains even in small confined environments such as farms. This is the first molecular epidemiology study of intermediately virulent <I>R. equi</I> isolates from Slovenian pigs.

Pulsed-field gel electrophoresis (PFGE) analysis of Listeria monocytogenes isolates from different sources and geographical origins and representative of the twelve serovars

Multiplex-PCR (MPCR) serogrouping and pulsed-field gel electrophoresis (PFGE) subtyping analysis are currently used by several public and private laboratories for the characterization of Listeria monocytogenes. In this study a set of 80 L. monocytogenes isolates belonging to the twelve serovars was used to investigate (i) the typeability of the rare serovars, (ii) the ability of PFGE analysis with ApaI and AscI to differentiate serovars within MPCR serogroups and (iii) the association of molecular types with the specific source or geographical origin of the isolates. With the exception of three isolates (rare serovars 4a and 4c) that were not amenable to restriction with ApaI, all the other analyzed isolates were subtyped by both enzymes. PFGE discriminated the 80 isolates into 62 combined ApaI and AscI PFGE patterns (pulsotypes), but could not differentiate serovars within MPCR serogroups, in which isolates from different serovars displaying the same pulsotype were found. Clustering analysis suggests that for some pulsotypes grouping according to Portuguese origin or source can be suggested. On the other hand, some L. monocytogenes clones are widely distributed. Two pulsotypes from Portuguese human isolates were identical to the ones displayed by human outbreak clones in the UK and in the USA and Switzerland, respectively, although they were not temporally matched. Computer-assisted data analysis of large and diverse PFGE type databases will improve the correct interpretation of subtyping data in epidemiological studies and in tracing routes and sources of contamination in the food industry.